LOS USOS DE ALFALTO NATURAL

Several antioxidant compounds were identified in primary screening, including ellagic acid, caffeic acid phenethyl ester, 7,4-dihydroxyflavone, and 7,2-dihydroxyflavone. Ellagic acid was of particular interest as it was the strongest hit in primary screening and it continued to perform well in follow-up studies (Figure 3.2 and Figure 3.3). In order to follow-up on the success of ellagic acid as an enhancer, other compounds with similar functionality were examined. Caffeic acid, an antioxidant which demonstrated some enhancement during primary screening, showed poor performance in a dose-response curve (Figure 3.4a). Ascorbic acid (vitamin C) was added to growth media, but no enhancement of viral transduction was observed (Figure 3.5). N-acetyl cysteine was also added with no enhancement of viral transduction (Figure 3.6). Other anti-oxidants

49 including beta-carotene and bilirubin were also examined, but poor solubility in DMSO or aqueous solution prevented their experimental use (data not shown).

Although ellagic acid is primarily considered as an antioxidant compound, it also serves as a tyrosine protein kinase inhibitor, can induce cell cycle arrest, and has intercalating properties.72-75 Due to the inability of other antioxidant compounds to facilitate adeno- associated virus transduction, it appears that the enhancement effect of ellagic acid is likely due to other effects.

3.4.4.1.

Ellagic Acid

Ellagic acid was the top hit in primary screening and continued to perform well in dose- response studies (Figure 3.2a). Ellagic acid showed an impressive level of enhancement in high throughput screening, and it continued to show dose-dependent enhancement during follow-up studies. However, ellagic acid also demonstrated limitations, primarily in solubility and toxicity. Dissolution of ellagic acid at concentrations up to 10 mM in either DMSO or aqueous solution was extremely challenging, and most studies were performed by dissolving ellagic acid directly in media and replacing the growth media in the well with filtered growth media containing ellagic acid at the required concentration. Differences in enhancement and toxicity between experimental runs may have been due to variability caused by the difficulty in dissolving ellagic acid at the required concentration.

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a)

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c)

Figure 3.2 Ellagic acid luciferase assay and viability results. a) Ellagic acid was dissolved at a concentration of 100 µM in media, serially diluted in media, and added to HAEC. 24 hours later, adeno-associated virus was added, followed by luciferase assay after another 24 hours. b) Normalized cell viability following 48 hours of cell exposure to ellagic acid dissolved in media. c) AAV serotype 2, 2/1, and 2/9 were added to HAEC following treatment with ellagic acid.

HAEC treated with 10 µM ellagic acid were additionally subject to transduction with AAV2-EGFP and examined using fluorescence microscopy, PCR, qRT-PCR, flow cytometry, and BCA assay. As shown in Table 3.1, the amount of viral DNA measured in the cell was the same as the 1% DMSO control, whereas viral RNA and fluorescence was greatly increased (61 fold change and 33 fold change, respectively). The total protein per well as measured by BCA assay increased two-fold.

Because the viral RNA and fluorescence levels changed so dramatically, the PCR data was examined more closely for insight into mechanism to determine whether this result

52 was a global increase in protein level or restricted to the increase in viral RNA and reporter gene signal (Table 3.2). The increase in GAPDH RNA and the large normalized GAPDH RNA/GAPDH DNA value, in combination with the two-fold increase in protein quantity by BCA assay, suggests that the results may be related to a global increase in overall protein. A previous report also described an increase in GAPDH mRNA and total protein levels as a result of ellagic acid exposure.75

GAPDH DNA GAPDH RNA (from cDNA) GAPDH RNA/ GAPDH DNA 1% DMSO* 1.0 1.0 1.0 Ellagic acid* 0.8 8.3 9.7 Tyrphostin 698* 3.3 0.4 0.1 Melphalan* 0.9 0.01 0.02 Vidarabine 4.3 0.1 0.03 5-bromo-2'- deoxyuridine 2.2 0.3 0.1 1,10-phenanthroline 0.7 0.1 0.1 Carboplatin 1.1 0.8 0.7

MOI 0 (no virus) 4.3 0.7 0.2

MOI 1,000 1.9 1.1 0.6

MOI 10,000 1.0 1.0 1.0

MOI 100,000 1.3 0.1 0.1

Table 3.2 Normalized values for quantitative real time PCR measurement of GAPDH DNA, GAPDH RNA (by measurement of cDNA), and the ratio of RNA/DNA. Compounds indicated with an asterisk have been normalized by the 1% DMSO measurement, whereas others were normalized by the MOI 10,000 condition.

Figure 3.3 provides corresponding information for fluorescence microscopy and flow cytometry. As compared with the 1% DMSO control, the cells treated with ellagic acid are elongated and much more fluorescent (Figure 3.3a,b). Flow cytometry showed more granularity in ellagic acid treated cells, as indicated by an increased side scatter measurement (Figure 3.3c,d). An overlay of the fluorescence clearly shows that the

53 ellagic acid treated cells have much higher levels of fluorescence following transduction with AAV2-EGFP (Figure 3.3e).

Figure 3.3. Fluorescence microscopy images and flow cytometry data for HAEC treated with ellagic acid followed by AAV2-EGFP transduction. a. Image of HAEC treated with 1% DMSO followed by AAV2-EGFP. b. Image of HAEC treated with ellagic acid in 1% DMSO followed by AAV2-EGFP. c. Forward and side scatter results for HAEC treated with 1% DMSO followed by AAV2-EGFP from flow cytometry. d.

54 Forward and side scatter results for HAEC treated with ellagic acid in 1% DMSO followed by AAV2-EGFP from flow cytometry. e. Histogram overlay of fluorescence area for HAEC treated with 1% DMSO (black) and ellagic acid in 1% DMSO followed by HAEC treated with AAV2-EGFP (red).

3.4.4.2.

Caffeic Acid Phenethyl Ester

Caffeic acid demonstrated a 2.5 fold enhancement in primary screening (Appendix Table A4) and was therefore examined more closely for a dose response. Additionally, caffeic acid phenethyl ester is an antioxidant compound, which allowed further probing of the potential for antioxidants to serve as a class of enhancing molecules.76 Figure 3.4a

below shows that the compound only moderately benefited viral transduction, while

Figure 3.4b demonstrates that cell viability was unaffected by the virus. a)

55 b)

Figure 3.4. Caffeic acid phenethyl ester results. a) Caffeic acid phenethyl ester was dissolved at a concentration of 10 mM in DMSO, serially diluted in DMSO, and added to HAEC cells in media such that the final DMSO concentration in each well was 1%. 24 hours later, adeno-associated virus was added, followed by luciferase assay after another 24 hours. b) Viability of HAEC following 48 hours of exposure to caffeic acid dissolved in DMSO.

3.4.4.3.

Ascorbic acid

In order to further examine antioxidant functions, ascorbic acid (vitamin C) was added to cells followed by treatment with virus containing the luciferase reporter gene. As shown in Figure 3.5 below, the addition of ascorbic acid had no effect on viral transduction.

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Figure 3.5. Ascorbic acid luciferase assay dose response results. Ascorbic acid was dissolved at a concentration of 100 µM in media, serially diluted in media, and added to HAEC. 24 hours later, adeno-associated virus was added, followed by luciferase assay after another 24 hours.

3.4.4.4.

N-acetyl cysteine

N-acetyl cysteine is another antioxidant compound.77 It was additionally tested in order to probe possible antioxidant enhancement of AAV2 transduction. The compound appears to have a moderate effect at best, most likely explained by an artifact of the experimental procedure (edge effects), as shown in Figure 3.6.

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Figure 3.6. N-acetyl cysteine luciferase assay dose-response results. N-acetyl cysteine was dissolved at a concentration of 100 µM in media, serially diluted in media, and added to HAEC. 24 hours later, adeno-associated virus was added, followed by luciferase assay after another 24 hours.